Thymidylate Synthase Inhibitor Research Articles

Abstract 3141: Combined cetuximab and pemetrexed therapy enhances cytotoxicity against crizotinib-resistant non-small cell lung cancer cells by downregulating thymidylate synthase

Abstract Although non-small cell lung cancer (NSCLC) cells with anaplastic lymphoma kinase (ALK)-rearranged initially show a dramatic response to crizotinib, these cells eventually develop resistance to crizotinib. This resistance is caused by secondary mutations and copy number gain in the ALK gene. However, other resistance mechanisms through activation of the bypass tracts have yet to be clearly elucidated. To investigate the mechanisms of acquired resistance to ALK fusion-directed treatment in NSCLC, we generated crizotinib-resistant NSCLC cell lines in vitro through chronic exposure of an ALK fusion NSCLC line (SNU2292) to crizotinib. Interestingly, the resultant SNU2292-CR cells maintained activation of epidermal growth factor receptor (EGFR) and expressed increased levels of transforming growth factor alpha (TGF-α), an EGFR ligand. Additionally, we found that thymidylate synthase (TS) was overexpressed in SNU2292-CR cells compared with the parental cells. We showed reduction of TS enhanced synergistic inhibition of cell proliferation when cetuximab, an EGFR inhibitor, was combined with pemetrexed, a TS inhibitor. As a result, combined therapy was found to exhibit a synergistic growth inhibitory effect against crizotinib-resistant cells by downregulating TS expression. Taken together, these results support a potential role of activation of the bypass tracts in acquired resistance to ALK-directed treatment in ALK-rearranged NSCLC, and provide insights into strategies for preventing and/or overcoming this resistance in patients Citation Format: Hwang-Phill Kim, Sae-Won Han, Sang-Hyun Song, Tae-You Kim. Combined cetuximab and pemetrexed therapy enhances cytotoxicity against crizotinib-resistant non-small cell lung cancer cells by downregulating thymidylate synthase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3141. doi:10.1158/1538-7445.AM2017-3141

Abstract 3990: Use of 3D tumoroid systems to define immune and cytotoxic therapeutic responses based on tumoroid and tissue slice culture molecular signatures

Abstract Historically, the successful establishment of exponentially growing viable in vitro tumor cell cultures has been found to occur from a minority of tumor tissues following artificial selection of a sub-population of tumor cells. Such limitations make it difficult to model patient variability in drug responses in vitro. Moreover, the lack of stromal cells that play a critical role in tumor viability, growth and drug response in vivo make the prediction of tumor response of immune-based therapeutics challenging. Through recent advances in three-dimensional (3D) culture (“tumoroid”) methodologies some of these hurdles are beginning to be addressed. However, it is currently unclear to what extent such models can be engineered to retain important phenotypic properties of infiltrating immune cells from the tumor tissue of origin. To better understand these limitations, we have developed 3D tumoroids and tumor slice in vitro cultures from tumors derived from patients with colorectal cancer (CRC) to evaluate immune cell populations infiltrating cultured CRC tissues. Furthermore, we have developed a system whereby the patient’s immune cells are re-incorporated into tumoroid in vitro cultures to evaluate the ability of the culture to mimic an immunosuppressive tumor microenvironment (ITM). Here we demonstrate the development of a propagating 3D epithelial tumoroid culture system from resected CRC tissue where we assessed the response to standard FDA approved therapy within weeks of surgical resection. Interestingly, tumoroid cultures from a CRC patient were highly sensitive to the thymidylate synthase inhibitor fluorouracil (adrucil) but less sensitive to the combination of the nucleoside analog trifluridine and the thymidine phosphorylase inhibitor tipiracil (Lonsurf). We isolated approximately four (4) million immune cells using Percoll density gradient centrifugation and flow cytometry. Re-introduction of isolated immune cells derived from surrounding and infiltrating tumor tissue and CD45+ tumor infiltrating hematopoietic cells displayed prolonged (>10 days) survival in co-culture. Moreover, established tumor slice cultures contained both an outer epithelial and inner stromal cell compartment mimicking tumor structure in vivo. Collectively, these data suggest that CRC tumoroid in vitro assays can be used for the assessment of some therapeutic responses. While further work is required to optimize this system, 3D tumoroid and slice culture assays may represent a novel in vitro approach to assess therapeutic efficacy of novel therapeutics and evaluate mechanisms of therapy resistance in general as well as for patient-specific response. Citation Format: Niklas K. Finnberg, Prashanth Gokare, Avital Lev, Alexander W. MacFarlane, Kerry S. Campbell, Karen Kaputa, Jeffrey Farma, Luigi Grasso, Nicholas C. Nicolaides, Wafik El-Deiry. Use of 3D tumoroid systems to define immune and cytotoxic therapeutic responses based on tumoroid and tissue slice culture molecular signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3990. doi:10.1158/1538-7445.AM2017-3990

Effective encapsulation and biological activity of phosphorylated chemotherapeutics in calcium phosphosilicate nanoparticles for the treatment of pancreatic cancer

Drug resistant cancers like pancreatic ductal adenocarcinoma (PDAC) are difficult to treat, and nanoparticle drug delivery systems can overcome some of the limitations of conventional systemic chemotherapy. In this study, we demonstrate that FdUMP and dFdCMP, the bioactive, phosphorylated metabolites of the chemotherapy drugs 5-FU and gemcitabine, can be encapsulated into calcium phosphosilicate nanoparticles (CPSNPs). The non-phosphorylated drug analogs were not well encapsulated by CPSNPs, suggesting the phosphate modification is essential for effective encapsulation. In vitro proliferation assays, cell cycle analyses and/or thymidylate synthase inhibition assays verified that CPSNP-encapsulated phospho-drugs retained biological activity. Analysis of orthotopic tumors from mice treated systemically with tumor-targeted FdUMP-CPSNPs confirmed the in vivo up take of these particles by PDAC tumor cells and release of active drug cargos intracellularly. These findings demonstrate a novel methodology to efficiently encapsulate chemotherapeutic agents into the CPSNPs and to effectively deliver them to pancreatic tumor cells.

5FU resistance caused by reduced fluoro-deoxyuridine monophosphate and its reversal using deoxyuridine.

The mechanism of 5-fluorouracil (5FU) resistance was investigated, focusing on the level of thymidylate synthase (TS) ternary complex formed with fluoro-deoxyuridine monophosphate (FdUMP). MKN45 and 5FU-resistant MKN45/F2R cells were treated with 5FU and fluoro-deoxyuridine (FdU) in combination with deoxyuridine (dU) and thymidine (dT). Subsequently, the levels of ternary complex were determined by western blotting and the cell viability was calculated using an MTT assay. MKN45/F2R cells exhibited 5FU resistance (56.2-fold relative to MKN45 cells), and demonstrated decreased orotate phosphoribosyltransferase (OPRT) and increased TS levels, requiring a higher concentration of 5FU to induce ternary complex formation than MKN45 cells. Following transfection of small interfering RNA against OPRT, MKN45 exhibited increased resistance to 5FU and decreased ternary complex formation subsequent to treatment with 5FU, indicating that decreased OPRT led to increased 5FU resistance. However, MKN45/F2R also exhibited resistance to FdU, which can be converted to FdUMP without OPRT, and there was decreased ternary complex formation after treatment with FdU, indicating that the 5FU-resistant cells had the ability to decrease intracellular FdUMP. The addition of dU and thymidine dT to 5FU promoted the formation of ternary complexes and reversed 5FU resistance in MKN45/F2R cells, although dT inhibited the efficacy of raltitrexed (another TS inhibitor). These results suggested that 5FU-resistant cells had the ability to reduce intracellular FdUMP irrespective of decreased OPRT, which led to resistance to 5FU. This resistance was then inhibited by treatment with dT or dU.

A simple and fast LC-MS/MS method with a very high sensitivity for the measurement of raltitrexed in human plasma.

Raltitrexed is a thymidylate synthase inhibitor that can be administered safely to patients with cardiovascular disease or dihydropyrimidine dehydrogenase deficiency, as opposed to 5FU. The recommended dose of 3mg/m2 every 3 weeks often leads to toxicity. Interestingly, the 2mg/m2 every 2 weeks dose appears to be less toxic. A pharmacokinetic trial was then performed by our team to investigate such phenomenon. However, there are currently, two main methods for RTX measurement described in the literature: a radioimmunoassay (RIA) and chromatographic-based methods with either UV or mass spectrometry detections. The RIA methods: display a low limit of quantification (below 1μg/L), but also a low extent of linearity for the calibration curve. The chromatographic-based methods: include high level of calibrators, but have poor sensitivity (>2μg/mL). If a high sensitivity is essential to satisfactorily describe the elimination of RTX, high concentrations in the calibration curve are also needed to avoid bias linked to the dilutions of the samples. A new LC-MS/MS method was then developed that allows to simultaneously measure very low (0.1μg/L) and very high (3000μg/L) concentrations in the same run. Moreover, the extraction steps are very simple and fast with mainly a precipitation and a filtration steps. This method was validated following the EMA recommendations. In view of the extent of the calibration curve, the carry-over effect was more deeply investigated. With this method, it was possible to measure RTX in samples taken 3 weeks after the administration. Taken together, this method allows to simply and quickly measure RTX in plasma of patients.

Dual addressing of thymidine synthesis pathways for effective targeting of proliferating melanoma.

Here, we examined the potential of blocking the thymidine de novo synthesis pathways for sensitizing melanoma cells to the nucleoside salvage pathway targeting endogenous DNA irradiation. Expression of key nucleotide synthesis and proliferation enzymes thymidylate synthase (TS) and thymidine kinase 1 (TK1) was evaluated in differentiated (MITF high [microphthalmia‐associated transcription factor] IGR1) and invasive (MITF medium IGR37) melanoma cells. For inhibition of de novo pathways cells were incubated either with an irreversible TS inhibitor 5‐fluoro‐2′‐deoxyuridine (FdUrd) or with a competitive dihydrofolate‐reductase (DHFR) inhibitor methotrexate (MTX). Salvage pathway was addressed by irradiation‐emitting thymidine analog [123/125I]‐5‐iodo‐4′‐thio‐2′‐deoxyuridine (123/125I‐ITdU). The in vivo targeting efficiency was visualized by single‐photon emission computed tomography. Pretreatment with FdUrd strongly increased the cellular uptake and the DNA incorporation of 125I‐ITdU into the mitotically active IGR37 cells. This effect was less pronounced in the differentiated IGR1 cells. In vivo, inhibition of TS led to a high and preferential accumulation of 123I‐ITdU in tumor tissue. This preclinical study presents profound rationale for development of therapeutic approach by highly efficient and selective radioactive targeting one of the crucial salvage pathways in melanomas.

An investigator-initiated phase I study of ONX-0801, a first-in-class alpha folate receptor targeted, small molecule thymidylate synthase inhibitor in solid tumors.

2503 Background: ONX-0801 is a first-in-class alpha folate receptor (AFR) targeted thymidylate synthase inhibitor, engineered to differentially accumulate 6000-fold in AFR overexpressing cancer cells. Methods: A 3+3 dose escalation design was used and two IV schedules were explored. Schedule A, weekly dosing (QW) and schedule B, once every 2 weeks dosing (Q2W). A cycle consisted of 4 weeks and treatment was stopped after 6 cycles in both schedules. An expansion cohort to evaluate clinical activity in patients with AFR overexpressing high grade serous ovarian cancer (HGSOC) was planned. Results: 21 patients each were treated in schedule A and B exploring doses ranging from 1-6 mg/m2 and 2-12 mg/m2, respectively. The dose limiting toxicity on schedule A was G3 cellulitis; no dose limiting toxicity was seen on schedule B. The most common toxicities were fatigue 15/42 (36%), nausea 9/42 (21%) and dysgeusia 5/42 (12%). Within schedule A at 4 mg/m2, 2 patients developed suspected drug-related changes on pulmonary function tests (drop in Dlco > 15%) at cycles 5 and 6, respectively. No cases of suspected drug-related drop in Dlco were noted in patients treated in schedule B. No grade 3-4 diarrhea, mucositis or neutropenia were seen in either cohort. The Cmax, AUC and half-life at 12 mg/m2 were 4952 ng/mL, 85170 h*ng/mL and 26 h, respectively. Pre-clinical PK-PD modelling aimed to achieve concentrations between 0.05-1 µM and this was achieved for periods of 48 h at doses of 4 mg/m2and above. Based on safety and PK, the recommended phase II dose (RP2D) of ONX-0801 was 12 mg/m2 Q2W and an expansion in patients with HGSOC is ongoing. 5 patients with HGSOC had partial responses (PRs) in the dose escalation cohort. In the current expansion cohort in patients with HGSOC, 5/11 patients had PRs. Archival samples have been analyzed from 8/11 patients in the expansion cohort. 4/4 AFR+ve and 4/4 AFR-ve patients did and did not have a PR following treatment with ONX-0801, respectively. Conclusions: The RP2D of ONX-0801 is 12 mg/m2 Q2W. At the RP2D, multiple patients with AFR overexpressing HGSOC have had PRs and further randomized biomarker prespecified phase II trials are warranted. Clinical trial information: NCT02360345.

A potential role of pemetrexed (PMTX) for the treatment (Tx) of thymidylate synthase (TS) negative primary brain tumors and systemic malignancies with CNS metastasis.

e13519 Background: Fluoropyrimidne analogue Capecitabine, a TS inhibitor, can cross blood brain barrier and has been used for the Tx of breast cancer with brain metastasis (mets). An antifolate Methotrexate (MTX), which also inhibits TS, has been used for the Tx of PCNSL secondary to it's excellent CNS penetration. MTX analogue, PMTX inhibits purine synthesis via TS and DHFR, and pyrimidine synthesis via GARFT, thus, making it a viable Tx option for CNS malignancies. Methods: We have treated 40 pts with previously heavily treated recurrent primary and systemic malignancies with CNS mets based on the molecular profiling. 26/40 pts were found to have TS negative expression (5/12 GBM, 5/5 AA, 1/1 Gr II Astrocytoma (A), 5/7 meningioma, 1/3 PCNSL, 1/2 Esthesioneuroblastoma, 1/1 chordoma, and 7/9 brain mets) and 10/26 TS negative pts were treated with PMTX. MRI was performed every 2-3 mo for tumor response. Results: Total pts: 10. M:F 6:4. Age: 48-88 yo. Median age: 61.9 yo. Tx period: 7/14–1/17. 1/1 GBM pt: stable disease (SD) x 4 mo. 1/2 AA pts: SD x 4 mo, 1/2 pts: tumor progression. 1 Gr. II A pt, SD x 7 mo. 2/2 meningioma pts: SD x 2 and 4 mo. Both pts also received concurrent cisplatin (also ERCC1 negative). 3/3 pts: squamous cell Ca of skin with skull base/brain mets responded x 24 mo, 24 mo and 3 mo respectively (1 PR and 2 SD), 2 pts: still receiving Tx. 1 pt: 26 mo reponder, died of nontumor related issue. 1/1 chordoma pt: SD x 11 mo. Toxicities: very mild except 2 pts (Gr. 3-4) who also received cisplatin. Conclusions: TS negative expression is commonly found in most of secondary malignancies with brain mets and in non-GBM primary brain tumors. PMTX appears to have a favorable efficacy for the Tx of TS negative primary and systemic malignancies with CNS mets. Albeit small cohort, overall response was 90% (PR:1, SD:8). Tx was particularly effective for squamous cell ca with brain mets and responses were durable in all spectrum. Due to favorable tolerance and response, especially in the elderly pts with squamous cell with brain mets, PMTX could be an excellent Tx option for TS negative CNS malignancies. PMTX trial for TS negative CNS malignancies should be considered.

Patent highlights December 2016-January 2017.

Patent highlights December 2016-January 2017.

The Role of Fluoropirimidines in Gastrointestinal Tumours: from the Bench to the Bed.

Gastrointestinal tumours are one of the most common types of cancer. Therapeutic options include surgery, radiotherapy, local ablation techniques, targeted agents, and chemotherapy. Fluoroprimidines are one of the most active drug families in digestive tumours and remains the cornerstone of the most commonly used chemotherapy schemes. We review the molecular basis of thymidylate synthase inhibition and the mechanisms of action of 5-fluorouracil, next generation oral fluoropyrimidines (capecitabine, tegafur and the latest S-1 and TAS-102) and antifolates. In addition, mechanisms and biomarkers of resistance and toxicity are explored. Finally, new fluoropyrimidines development and clinical trials ongoing in digestive tumours are reviewed. Further research is necessary to avoid resistance mechanisms, improve clinical outcomes and continue reducing toxicities. Until new drugs become available, the optimization of current therapies should be a priority.

The Possible Cardio-Protective Effects of Ethanolic Artichoke Extract against 5- Fluorouracil Induced Cardiac Toxicity in Rats

Cardiac toxicity can occur during the therapy with several cytotoxic drugs, including 5- Fluorouracil (5- FU). It is an antimetabolite that acts during the S phase of the cell cycle and is activated by thymidine phosphorylase into fluorodeoxyuridylate (5 fluoro 2'deoxyuridine 5'monophosphate, 5-FdUMP) that inhibits thymidylate synthase, thus preventing DNA synthesis that leads to imbalanced cell growth and ultimately cell death. It is still a widely used anticancer drug, since 1957. The present study aimed to evaluate the possible cardio-protective effects of ethanolic artichoke extract (Cynara scolymus L.) against 5-fluorouracil (5-FU) induced cardio-toxicity in rats by evaluating serum levels of Alanine aminotransferase, aspartate aminotransferase and creatine kinase enzymes. Methods: Twenty -four female albino rats were randomly divided into 4 groups each group with 6 rats. Group I: (negative control) received oral daily dose of dimethyl sulfoxide (DMSO) (2 ml/kg /day) for 10 successive days. Group II: (positive control) received oral daily dose of DMSO (2 ml/kg /day) for 10 successive days and subsequently administered single dose of 5-FU (150 mg/kg) by intraperitoneal injection on 8th day in association with DMSO. Groups III: received oral daily dose of ethanolic artichoke extract (200 mg/kg/day) for 10 successive days. Groups IV: received oral daily dose of ethanolic artichoke extract (200 mg/kg/day) for 10 successive days with subsequently administered single intraperitoneal dose of 5-FU (150 mg/kg) on 8th day in association with ethanolic extract. Results: Treatment of ethanolic artichoke extract prior 5-FU intoxication significantly attenuate the increase of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatine kinase (CK) enzymes activities caused by 5-FU-induced cardio-toxicity in rats. Conclusions: Results of the present finding suggest that the ethanolic artichoke extract may be an effective modulator in mitigating 5-FU induced cardiac toxicity in rats.Keywords: Ethanolic artichoke extract, 5-Fluorouracil, Cardio-protection, AST, ALT and CK.

Synthesis and evaluation of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines as receptor tyrosine kinase and thymidylate synthase inhibitors and as antitumor agents

In an effort to optimize the structural requirements for combined cytostatic and cytotoxic effects in single agents, a series of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines 3–7 were synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs) as well as thymidylate synthase (TS). The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-bromo/5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate aryl thiols. A novel four step synthetic scheme to the common intermediate was developed which is more efficient relative to the previously reported six-step sequence. Biological evaluation of these compounds indicated dual activity in RTKs and human TS (hTS). In the VEGFR-2 assay, compound 5 was equipotent to the standard compound semaxanib and was better than standard TS inhibitor pemetrexed, in the hTS assay. Compounds 3, 6 and 7 were nanomolar inhibitors of hTS and were several fold better than pemetrexed.

5-fluorouracil Toxicity Mechanism Determination in Human Keratinocytes: in vitro Study on HaCaT Cell Line.

5-fluorouracil (5-FU) and capecitabine therapy is often accompanied by palmar-plantar erythrodysesthesia (PPE) which is manifestation of 5-FU toxicity in keratinocytes. The main mechanisms of 5-FU action are thymidylate synthase (TS) inhibition which can be abrogated by thymidine and strengthened by calciumfolinate (CF) and incorporation of fluorouridinetriphosphate into RNA which can be abrogated by uridine. For proper PPE treatment 5-FU mechanism of action in keratinocytes needs to be elucidated. We used the 5-FU toxicity modulators uridine, thymidine and CF to discover the mechanism of 5-FU action in human keratinocyte cell line HaCaT. To measure the cellular viability, we used MTT test and RTCA test. CF did not augment 5-FU toxicity and 5-FU toxicity was weakened by uridine. Therefore, the primary mechanism of 5-FU toxicity in keratinocytes is 5-FU incorporation into RNA. The uridine protective effect cannot fully develop in the presence of CF. Thymidine addition to 5-FU and uridine treated cells not only prevents the toxicity-augmenting CF effect but it also prolongs the 5-FU treated cells survival in comparison to uridine only. Therefore, it can be assumed that in the presence of uridine the 5-FU toxicity mechanism is switched from RNA incorporation to TS inhibition. Although particular 5-FU toxicity mechanisms were previously described in various cell types, this is the first time when various combinations of pyrimidine nucleosides and CF were used for 5-FU toxicity mechanism elucidation in human keratinocytes. We suggest that for PPE treatment ointment containing uridine and thymidine should be further clinically tested.

P3.03-026 Orotate Phosphoribosyltransferase is Overexpressed in Malignant Pleural Mesothelioma: Dramatically Responds One Case in High OPRT Expression: Topic: Mesothelioma Transitional

Malignant pleural mesothelioma (MPM) is a rare and aggressive, treatment-resistant cancer. Pemetrexed, an inhibitor of thymidylate synthase (TS), is used worldwide for MPM as a first-line chemotherapy regimen. However, there is little consensus for a second-line chemotherapy. S-1, a highly effective dihydropyrimidine dehydrogenase (DPD)-inhibitory fluoropyrimidine, mainly acts via a TS inhibitory mechanism similar to pemetrexed. Orotate phosphoribosyltransferase (OPRT) is a key enzyme related to the first step activation of 5-fluorouracil (5-FU) for inhibiting RNA synthesis. We investigated 5-FU related-metabolism proteins, especially focusing on OPRT expression, in MPM. Fifteen MPM patients who were diagnosed between July 2004 and December 2013 were enrolled. We examined the protein levels of 5-FU metabolism-related enzymes (TS, DPD, OPRT, and thymidine phosphorylase [TP]) in 14 cases. High TS, DPD, OPRT, and TP expressions were seen in 28.6%, 71.4%, 85.7%, and 35.7% of patients, respectively. We found that OPRT expression was extremely high in MPM tissue. We experienced one remarkable case of highly effective S-1 combined therapy for pemetrexed refractory MPM. This case also showed high OPRT protein expression. The present study suggests that OPRT expression is high in MPM tumors. Although pemetrexed is mainly used for MPM chemotherapy as a TS inhibitor, S-1 has potential as an anticancer drug not only as a TS inhibitor but also inhibiting RNA synthesis through the OPRT pathway. This is the first report investigating OPRT protein expressions in MPM.

Gemcitabine Mechanism of Action Confounds Early Assessment of Treatment Response by 3'-Deoxy-3'-[18F]Fluorothymidine in Preclinical Models of Lung Cancer.

3'-Deoxy-3'-[18F]fluorothymidine positron emission tomography ([18F]FLT-PET) and diffusion-weighted MRI (DW-MRI) are promising approaches to monitor tumor therapy response. Here, we employed these two imaging modalities to evaluate the response of lung carcinoma xenografts in mice after gemcitabine therapy. Caliper measurements revealed that H1975 xenografts responded to gemcitabine treatment, whereas A549 growth was not affected. In both tumor models, uptake of [18F]FLT was significantly reduced 6 hours after drug administration. On the basis of the gemcitabine concentration and [18F]FLT excretion measured, this was presumably related to a direct competition of gemcitabine with the radiotracer for cellular uptake. On day 1 after therapy, [18F]FLT uptake was increased in both models, which was correlated with thymidine kinase 1 (TK1) expression. Two and 3 days after drug administration, [18F]FLT uptake as well as TK1 and Ki67 expression were unchanged. A reduction in [18F]FLT in the responsive H1975 xenografts could only be noted on day 5 of therapy. Changes in ADCmean in A549 xenografts 1 or 2 days after gemcitabine did not seem to be of therapy-related biological relevance as they were not related to cell death (assessed by caspase-3 IHC and cellular density) or tumor therapy response. Taken together, in these models, early changes of [18F]FLT uptake in tumors reflected mechanisms, such as competing gemcitabine uptake or gemcitabine-induced thymidylate synthase inhibition, and only reflected growth-inhibitory effects at a later time point. Hence, the time point for [18F]FLT-PET imaging of tumor response to gemcitabine is of crucial importance. Cancer Res; 76(24); 7096-105. ©2016 AACR.

Progression in the treatment of raltitrexed for colorectal cancer

Raltitrexed is a kind of specific thymidylate synthase inhibitor, and as a novel cytotoxic drug, it is used in the treatment of a variety of solid tumors, with the advantages of good effect, light adverse reaction and easy to use.Especially in the treatment of the colorectal cancer, its efficacy has been recognized.And in clinical, raltitrexed can plug with chemotherapy or targeted drug combination, as well as radiosensitizer.This review summarized the raltitrexed research progress on the treatment of colorectal cancer in recent years. Key words: Raltitrexed; 5-Fluorouracil; Tymidylate synthase; Colorectal cancer; Radiotherapy

Dual targeting of glutaminase 1 and thymidylate synthase elicits death synergistically in NSCLC.

Glutaminase 1 (GLS1) expression is increased in non-small cell lung cancer (NSCLC). GLS1 knockdown using siRNA or inhibition using bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) induced cell cycle arrest with significant reduction of ATP level while levels of reactive oxygen species or glutathione were not affected in NSCLC cell lines. Recently we found that NSCLC significantly depends on cytosol NADH for ATP production. GLS1 remarkably contributes to ATP production through transferring cytosolic NADH into mitochondria via malate-aspartate shuttle by supply of glutamate in NSCLC. Regulation of malate-aspartate shuttle by knockdown or inhibition of glutamic-oxaloacetic transaminase 2 or malate dehydrogenase 2 mimicked GLS1 knockdown, which induced cell death with ATP reduction in NSCLC. Therefore, GLS1 inhibition induced cell cycle arrest with ATP depletion by glutamate reduction. Dual inhibition with BPTES and thymidylate synthase inhibitor, 5-fluorouracil (5-FU), elicits cell death synergistically through cell cycle arrest in NSCLC. A preclinical xenograft model of NSCLC showed remarkable anti-tumour effect synergistically in the BPTES and 5-FU dual therapy group.

MPTH-58. PEMETREXED: A POTENTIAL NEW THERAPEUTIC OPTION FOR THE TREATMENT OF THYMIDYLATE SYNTHASE (TS) NEGATIVE RECURRENT PRIMARY AND SYSTEMIC MALIGNANCIES WITH CNS METASTASIS

Fluoropyrimidine analogues, 5FU, Capecitabine and Pemetrexate, inhibit thymidylate synthase and they have been widely used for the treatment of systemic malignancies. Capecitabine can penetrate CNS and has shown some efficacy in the treatment of breast cancers with CNS metastasis. The role of Pemetrexed in the treatment of recurrent primary and systemic malignancies with CNS metastsis is not known. We have treated 33 pts with previously heavily-treated recurrent primary and systemic malignancies with CNS metastasis based on the results of the molecular profiling. 16/33 pts were found to have TS-negative and 9/16 TS-negative patients were treated with Pemetrexed. MRI was performed every 2-3 months for tumor evaluation. Total pts: 9. M:F 5:4, Age: 48-88 yo. Median age 70.7 yo. Treatment period: 7/14 - 6/16. 1/1 GBM pt: SD for 4 mo. 1/2 anaplastic astrocytoma pts: SD for 4 mo. 1 Gr. II astrocytoma pt: SD for 7 mo. 2/2 meningioma pts: SD for 2 mo (still on treatment) and 4 mo. Both pts also received concurrent cisplatin based on ERCC1 and BRCA1 markers. 2/2 pts with squamous cell Ca of skin with skull base/brain metastasis: 1 PR for 24 mo, 1 SD for 18 mo respectively and still receiving treatment. 1 chordoma pt involving cervical/skull base: SD for 11 mo: still receiving treatment. Toxicities were very mild except 2 meningioma pts (Gr. 3-4) who also received cisplatin. Pemetrexed treatment for TS-negative recurrent primary and systemic malignancies with CNS metastasis demonstrated an excellent response rate of 89% (PR:1, SD:7, TP:1). Toxicities were minimal and tumor responses were durable. Particularly in the elderly population, in light of excellent toxicity profile, Pemetrexed should be considered as a part of the first treatment options for TS-negative CNS malignancies. A TS directed trial for pts with TS-negative recurrent CNS malignancies is warranted.

Early detection of pemetrexed-induced inhibition of thymidylate synthase in non-small cell lung cancer with FLT-PET imaging.

Inhibition of thymidylate synthase (TS) results in a transient flare in DNA thymidine salvage pathway activity measurable with FLT ([18F]thymidine)-positron emission tomography (PET). Here we characterize this imaging strategy for potential clinical translation in non-small cell lung cancer (NSCLC). Since pemetrexed acts by inhibiting TS, we defined the kinetics of increases in thymidine salvage pathway mediated by TS inhibition following treatment with pemetrexed in vitro. Next, using a mouse model of NSCLC, we validated the kinetics of the pemetrexed-mediated flare in thymidine salvage pathway activity in vivo using FLT-PET imaging. Finally, we translated our findings into a proof-of-principle clinical trial of FLT-PET in a human NSCLC patient. In NSCLC cells in vitro, we identified a burst in pemetrexed-mediated thymidine salvage pathway activity, assessed by 3H-thymidine assays, thymidine kinase 1 (TK1) expression, and equilibrative nucleoside transporter 1 (ENT1) mobilization to the cell membrane, that peaked at 2hrs. This 2hr time-point was also optimal for FLT-PET imaging of pemetrexed-mediated TS inhibition in murine xenograft tumors and was demonstrated to be feasible in a NSCLC patient. FLT-PET imaging of pemetrexed-induced TS inhibition is optimal at 2hrs from therapy start; this timing is feasible in human clinical trials.

The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine

Decitabine (5-aza-2'-deoxycytidine, aza-dCyd) is an anti-cancer drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukaemia that can act as a DNA-demethylating or genotoxic agent in a dose-dependent manner. On the other hand, DCTPP1 (dCTP pyrophosphatase 1) and dUTPase are two 'house-cleaning' nucleotidohydrolases involved in the elimination of non-canonical nucleotides. In the present study, we show that exposure of HeLa cells to decitabine up-regulates the expression of several pyrimidine metabolic enzymes including DCTPP1, dUTPase, dCMP deaminase and thymidylate synthase, thus suggesting their contribution to the cellular response to this anti-cancer nucleoside. We present several lines of evidence supporting that, in addition to the formation of aza-dCTP (5-aza-2'-deoxycytidine-5'-triphosphate), an alternative cytotoxic mechanism for decitabine may involve the formation of aza-dUMP, a potential thymidylate synthase inhibitor. Indeed, dUTPase or DCTPP1 down-regulation enhanced the cytotoxic effect of decitabine producing an accumulation of nucleoside triphosphates containing uracil as well as uracil misincorporation and double-strand breaks in genomic DNA. Moreover, DCTPP1 hydrolyses the triphosphate form of decitabine with similar kinetic efficiency to its natural substrate dCTP and prevents decitabine-induced global DNA demethylation. The data suggest that the nucleotidohydrolases DCTPP1 and dUTPase are factors involved in the mode of action of decitabine with potential value as enzymatic targets to improve decitabine-based chemotherapy.